JP2008007513A - Receptor-specific transepithelial transport of immunogen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a product for modulating a mammalian immune response. <P>SOLUTION: A pharmaceutical preparation is provided, containing a conjugate of an antigen and an FcRn binding partner. The conjugate can be administered transepithelially to mammals needing immune modulation. The conjugate is administered to mammals in effective amounts to modulate the immune system by stimulating the immune response against the antigen or tolerating the immune system to the antigen. The antigen may be characteristic of a pathogen, of an autoimmune disease, of an allergen or of a tumor. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

Detailed Description of the Invention

The present invention relates generally to methods and products for initiating an immune response against an antigen, and in particular to transepithelial delivery of an antigen that causes tolerance and immunity.

BACKGROUND OF THE INVENTION The mammalian immune system appears during pregnancy and becomes active in late mammalian fetuses. Although active, it may still be characterized as “mature” because it is hardly stimulated by the antigen, ie, the fetus is protected from the antigen primarily by the mother. However, this “immature” immune system is supplemented by transmission of maternal immunoglobulin to the fetus (or in some cases to the newborn), and the humoral immunity during the first weeks of life independently. give.

  Rats and mice obtain most maternal immunoglobulin G (IgG) while suckling from colostrum and milk, but to some extent prenatally. Cattle also obtain IgG from colostrum. In rabbits, IgG is transported through the yolk sac to the fetus. Little is known about the transfer of IgG to the fetus or neonate in humans. Many results suggest that human mothers transmit humoral immunity to their babies only before birth, but IgA transmitted to newborns by breast milk helps protect newborns from enteric infections. It is thought that he plays a role.

  Supplying maternal IgG to mammals and / or neonates requires transport across the epithelial barrier that is largely impermeable to macromolecules. Such transport of macromolecules across the epithelial barrier can be triggered by non-specific and specific receptor-dependent mechanisms. The receptor non-specific mechanism is shown by the paracellular sieving phenomenon, the efficiency of which is inversely related to the molecular weight of the molecule being transported. Transport of macromolecules such as IgG through this paracellular pathway is extremely inefficient. Description of receptor-dependent transport of immunoglobulins by intestinal epithelial cells has so far been based on polymer immunoglobulin receptors and IgG enterocyte receptors (major histocompatibility complex (MHC) class I-related Fc receptors Body). These two receptor systems differ in their immunoglobulin isotype, their direction of immunoglobulin transport through epithelial cells, and their tissue-specific expression. Both can play a role in forming an immature immune system.

  Polymeric immunoglobulin receptors are expressed on the basolateral surface of enterocytes, hepatocytes and / or bile duct epithelial cells. It transports the polymers IgA and IgM to the tip (lumen) surface, concentrating these immunoglobulins for antimicrobial defense and antigen exclusion.

Intestinal cell receptor of IgG, and beta ₂ has homology to the MHC class I heavy chain - optionally combined with microglobulin (β ₂ M), it is expressed in the rat and mouse neonatal enterocytes. IgG is transcellularly transported through the intestinal epithelium of these rodent neonates and through the lumen into the serosa. On the apical surface of the enterocytes, the Fc portion of IgG is bound to the enterocyte receptor at the relatively acidic pH of the lumen (about pH 6.0). After transcytosis of the basolateral plasma membrane, the release of immunoglobulin occurs at the relatively neutral pH of the interstitial fluid (about pH 7.4). Thus, the rodent neonatal Fc receptor (FcRn) could be involved in the supply of maternal IgG to the newborn and could itself be involved in the passive acquisition of IgG during this period.

  In humans, maternal IgG is actively transported across the placenta. The receptors involved in this transport have been explored for years. Several IgG binding proteins have been isolated from the placenta. FcγRII was detected in placental endothelium and FcγRIII was detected in syncytiotrophoblast layer. However, both of these receptors showed a relatively low affinity for monomeric IgG. Recently, the isolation from the placenta of a cDNA encoding the human homologous chromosome of rat and mouse IgG enterocyte receptors has been reported. (Story, C.M. et al., J. Exp. Med., 180: 2377-2381, December 1994) The complete nucleotide sequence and deduced amino acid sequence have been reported. This Fc receptor for IgG is highly homologous across its reading frame, including the rat FcRn sequence (69% nucleotide identity and 65% putative amino acid identity), so to the human fetus (and possibly even to the newborn) May be involved in the transport of maternal IgG. So-called passive immunization in human fetuses (and possibly human newborns) can now be better understood.

  In contrast to passive immunization, which requires supplementing the host's immune system with antibodies from other sources, active immunization involves the host's own immune system to produce the desired immune response in vivo. Requires irritation. The most widely practiced method of active immunization in children and adults involves injecting the immunogen once as an initial dose, followed by at least one additional booster dose. These methods have a number of significant drawbacks, including the risks associated with the use of needles that can transmit diseases such as AIDS and hepatitis. (If the patient is tolerated to allergens, the problem is exacerbated by the frequent need for repeated injections over time.) These methods also provide first-line protection against numerous pathogens, ie It does not necessarily cause mucosal immunity. The mucosa wraps inside the respiratory tract, reproductive system and gastrointestinal tract, and this mucosal surface is the first entry point for many diseases. An oral vaccine that is easy to supply and causes mucosal immunity would be highly desirable.

  Immunization using an oral vaccine is problematic. Often, little or no immune response is obtained. In order to promote an immune response, the antigen of interest has been bound to a carrier known to be highly immunogenic. For example, researchers have supplied antigens using Bacille Calmette-Gurein (BCG) as a carrier, and BCG is the first bacterium used as an oral vaccine against tuberculosis. The problem with such a carrier is that the patient will raise antibodies against the carrier itself, which can be cumbersome if the carrier is used again to give another antigen to the same patient. There can be. To date, the success of general strategies for oral vaccines has not been demonstrated.

  Traditionally, immunoglobulins and portions thereof have been attached to drugs and contrast agents to concentrate and destroy cell populations and to extend the half-life of some drugs. An immunotoxin is an example of such a conjugate. However, such conjugates have never been proposed as useful for initiating an immune response.

  A small amount of research has focused on the ability to produce immune tolerance of immunoglobulins bound to oligonucleotides or proteins characteristic of autoimmune diseases. (See PCT WO91 / 08773). This study is based on the notion that tolerance induction can be strongly influenced by carrier molecules and that immunoglolin carriers are likely to produce immune tolerance. Allogeneic syngeneic IgG is the preferred carrier, and intravenous administration was the method used to deliver the conjugate of IgG. Although this work has been over 10 years, oral administration is described only once and only for conjugates where IgA is an immunoglobulin carrier. Thus, immunoglobulin conjugates that produce immune tolerance are known in the art, but such conjugates have been suggested as agents to elicit strong responses to antigens specific to pathogens. There was no. (On the contrary, the art suggests that any such conjugate would tolerate the subject against pathogens that are considered highly undesirable). Furthermore, such conjugates have never been suggested to be an effective tolerogenic antigen when the immunoglobulin is IgG and the delivery system is oral delivery.

SUMMARY OF THE INVENTION The present invention binds an antigen to a molecule that binds to an FcRn receptor, such as an immunoglobulin or portion thereof, and crosses the epithelial barrier by active transport through the enterocytes by the FcRn receptor. It is related to the knowledge that can be supplied. The immunoglobulin or portion thereof binds to the FcRn receptor and serves as a carrier for the antigen so that the immunoglobulin or portion thereof is transported across the epithelial barrier by FcRn-dependent transport. FcRn receptors are present in epithelial tissues of human children and adults, and therefore the present invention allows an effective strategy for immunizing humans.

  In accordance with one embodiment of the present invention, a method for modulating the immune system of a mammal is provided. An effective amount of an antigen and FcRn binding partner conjugate is administered to the epithelial barrier of a mammal in need of such immunomodulation. The antigen is selected from the group consisting of an antigen exhibiting pathogen characteristics, an antigen exhibiting autoimmune disease characteristics, an antigen exhibiting allergen characteristics, and an antigen exhibiting tumor characteristics. In a preferred embodiment, the FcRn binding partner is non-specific IgG, or an FcRn binding fragment of IgG. Most preferably, the FcRn binding partner is an Fc fragment of IgG. Furthermore, it is preferred that the antigen is covalently bound to the FcRn binding partner. Preferably, the conjugate is administered orally to the intestinal epithelium, aerosol to the lung or intranasally. Such formulations may not be sterile. Supplementary enhancers as described below may be further administered.

  According to another aspect of the invention, a formulation is provided. The formulation comprises a conjugate of an antigen and an FcRn binding partner, the antigen comprising an antigen that is characteristic of a pathogen, an antigen that is characteristic of an autoimmune disease, an antigen that is characteristic of an allergen, and an antigen that is characteristic of a tumor Selected from the group. Preferred FcRn binding partners are as described above. The conjugate is present in an effective amount that modulates the mammalian immune response. The formulation further comprises a pharmaceutically acceptable carrier. The formulations of the present invention are formulated in unit dosage forms constructed and tailored for delivery to epithelial transport carriers such as oral delivery to the intestinal epithelium, aerosol delivery to the lung epithelium and intranasal delivery to the nasal epithelium. sell. Thus, tablets containing IgG (or its FcRn binding portion) bound to any of the antigens characterized above are encompassed by the present invention.

  The aforementioned formulations can be supplied with supplemental enhancers including adjuvants, cytokines, bioadhesives and the like. The supplemental enhancer itself can bind to the FcRn binding partner and facilitate the delivery of such agents across the epithelial barrier. Preferred modes of administration generally include oral dosing to the intestinal epithelium, aerosols to the lungs and intranasal dosing.

According to another embodiment of the present invention, the above conjugate is used in the manufacture of a medicament for modulating the mammalian immune system. Preferred ingredients and formulations are as described above.
According to yet another aspect of the invention, a method for producing an immunomodulatory agent is provided. The method involves covalently binding an antigen or supplement enhancer to an FcRn binding partner, wherein the antigen or supplement enhancer is selected as described above. Preferred FcRn binding partners are also as described above. The conjugate can then be used to produce a formulation for modulating a mammal's immune response as described above.

  In yet another embodiment of the invention, the conjugate comprising the antigen crosses the epithelial barrier in an amount at least twice the extent that the antigen crosses the epithelial barrier in an unbound form. Thus, an object of the present invention is to develop a mechanism that increases the ability of an antigen to cross the epithelial barrier.

Another object of the present invention is to develop a new class of active immunogens and tolerogenic antigens orally.
Another object of the present invention is to develop an improved method of stimulating mucosal immunity.

These and other aspects of the invention are described in further detail below.
Detailed Description of the Invention The present invention uses the finding that human FcRn receptors are active in adult epithelial tissue and FcRn binding partners such as IgG or Fc fragments to cross the epithelial barrier to other molecules, including antigens. It is related to the knowledge that can be transported. In this manner, FcRn binding partners, such as IgG or its FcRn binding portion, can be used to cross the epithelial barrier to supply antigen to the subject's immune system, thereby initiating an immune response.

  The present invention is useful whenever it is desired to facilitate the supply of antigens across the epithelial barrier to the immune system. Thus, the present invention can be used to deliver antigen across intestinal epithelial tissue, lung epithelial tissue, and other mucosal surfaces including nasal surface, vagina surface, colon surface and biliary dendritic surface. . The present invention can be used to modulate a subject's immune system, such as by stimulating a humoral antibody response to an antigen, stimulating T cell activity, or stimulating tolerance to an antigen. As used herein, subject refers to humans, primates, horses, cows, sheep, pigs, goats, dogs, cats, chickens and rodents.

  When supplying tumor antigens, the present invention can be used to treat subjects with diseases that follow immune-dependent rejection, such as small non-solid tumors or solid tumors. Furthermore, it is believed that the delivery of tumor antigens by the methods described herein will be useful for treatments involving removal of large solid tumors. The invention can further be used to treat a subject suspected of having cancer.

  The present invention relates to the formation of FcRn binding partners and antigen conjugates. Conjugates are those that bind to each other by any physicochemical means including hydrophobic interaction between the antigen and the non-specific hydrophobic portion of the antibody molecule, antibody-antigen specific binding, and covalent bonding. Means one substance. The preferred binding properties will depend in particular on the mode of administration and drug carrier used to deliver the conjugate to the selected epithelial barrier. For example, some bonds are not as well suited as others that withstand certain environments, such as the stomach, but can be protected therefrom by a delivery system that avoids the stomach. Of course, it is important that the binding between the FcRn binding partner and the antigen has the property that it does not destroy the ability of the FcRn binding partner to bind to the FcRn receptor. Such couplings are well known to those skilled in the art and examples are given in more detail below. The conjugate can also be formed as a fusion protein, discussed in more detail below.

  By FcRn binding partner is meant any substance that can specifically bind by the FcRn receptor with active transport resulting from the FcRn binding partner's FcRn receptor. As mentioned above, FcRn receptors have been isolated for several mammalian species, including humans. The sequences of human FcRn, rat FcRn and mouse FcRn can be found in Sutrie, C.M. et al., J. Exp. Med., 180: 2377-2381, December 1994. FcRn receptor molecules are well characterized here. The FcRn receptor binds IgG at a relatively low pH (rather than other immunoglobulin classes such as IgA, IgD, IgM and IgE), and actively binds the IgG in the serosa direction through the lumen through cell penetration After transport, it releases IgG at the relatively high pH found in intestinal fluid. As will be appreciated by those skilled in the art, FcRn receptors can be isolated by cloning or by affinity purification using, for example, monoclonal antibodies. Such isolated FcRn receptors can then be used to identify and isolate FcRn binding partners as described below.

FcRn binding partners include whole IgG, Fc fragments of IgG, and other fragments of IgG that contain the complete binding portion of the FcRn receptor. The range of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister, WP et al., Nature, 1994; 372: 379-378). The main contact site between Fc and FcRn receptor is near the junction of C _H 2 and C _H 3 domains. Contactable are 385～387,428 and 433-436 in C _H 2 residues 248,250～257,272,285,288,290～291,308～311 and 314 and C _H 3. (These sites are different from those identified by subclass comparisons or by site-directed mutagenesis as is important for Fc binding to leukocytes FcγRI and FcγRII.) All the Fc-FcRn contacts described above are single. In the Ig heavy chain. It has previously been noted that two FcRn receptors can bind one Fc molecule. Crystallographic data suggest that in such a complex, each FcRn molecule binds one polypeptide of the Fc homodimer.

  Given the above information, those skilled in the art will modify the Fc portion of IgG according to well-understood procedures such as site-directed mutagenesis and the like, which will be bound by FcRn receptors It will be readily appreciated that modified IgGs or modified Fc fragments or portions thereof can be generated. Such modifications include modifications remote from the FcRn contact site as well as modifications in the contact site that may preserve or even enhance binding. In addition, other binding partners can be identified and isolated. Antibodies or portions thereof that are specific for the FcRn receptor and can be transported by FcRn once bound can be identified and isolated using well-established techniques. Similarly, randomly generated molecularly distinct libraries can be screened, and molecules bound and transported by FcRn receptors can be isolated using conventional techniques. . The present invention is not limited by the choice of any particular FcRn binding partner. If the binding partner is IgG or an FcRn binding portion thereof, the IgG or portion thereof can be prepared according to conventional methods described in further detail below.

  An FcRn binding partner is bound to the antigen. Antigens used herein are divided into four classes: (1) antigens that are characteristic of pathogens; (2) antigens that are characteristic of autoimmune diseases; (3) antigens that are characteristic of allergens; and (4) Divided into antigens that show the characteristics of the tumor. Antigens generally include polysaccharides, glycolipids, glycoproteins, peptides, proteins, carbohydrates and lipids from the cell surface, cytoplasm, nucleus, mitochondria and the like.

  Antigens that are characteristic of pathogens include antigens derived from viruses, bacteria, parasites or fungi. Examples of important pathogens include Vibrio cholerae, Escherichia coli, rotavirus, Clostridium difficile, Shigella species, Salmonella typhi , Parainfluenza virus, influenza virus, Streptococcus pneumoniae, Borella burgdorferi, HIV, Streptococcus mutans, Plasmodium falciparum, Staphylococcus aureus ( Staphylococcus aureus), rabies virus and Epstein-Barr virus.

  Viruses are generally not limited to the following families: Picornaviridae; Caliciviridae; Togaviridae; Flaviviridae; Coronaviridae; Rhabdoviridae; Filoviridae; Paramyxoviridae; Orthomyxoviridae; Bunyaviridae; Arenaviridae; Reoviridae; Retroviridae; Hepadonaviridae; Parvoviridae; Papovaviridae; Adenoviridae; Herpesviridae; There is Poxyviridae.

  Bacteria generally include, but are not limited to, P. aeruginosa; E. coli; Klebsiella species; Serratia species; Pseudomonas species; P. cepacia ( cepacia); Acinetobacter species; S. epidermis; E. faecalis; Streptococcus pneumoniae; Staphylococcus aureus; Haemophilus species; Neisseria species; N. meningitidis; Bacteroides species; Citrobacter species; Branhamella species; Salmonella species; Shigella species; S. pyogenes; Proteus Proteus species; Clostridium species; Erysipelothrix species; Lesteria species; Pasteurella mar Pasteurella multocida; Streptobacillus species; Spirillum species; Fusospirocheta species; Treponema pallidium; Borrelia species; Actinomycetes; Actinomycetes Mycoplasma species; Chlamydia species; Rickettsia species; Spirochaeta; Legionella species; Mycobacteria species; Ureaplasma species; Streptomyces species (Streptomyces) species; Trichomoras species; and P. mirabilis.

  Parasites include, but are not limited to, Plasmodium falciparum, Plasmodium falciparum (P. vivax), Oval malaria parasite (P. ovale), Plasmodium falciparum (P. malaria); Toxoplasma gondii; Leishmania mexicana, L. tropica, L. major, L. major, aethiopica, L. donovani; Trypanosoma cruzi ( Trypanosoma cruzi, T. brucei; Schistosoma mansoni, S. haematobium, S. japonium; Trichinella spiralis; Wancroft bancrofti); Brugia malayi; Entamoeba histolytica; Enterobius vermiculoarus; Taenia solium; T. sagina ta); Trichomonas vaginatis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidium parvum; Pneumocystis carinii); Babesia bovis, B. divergens, B. microti; Isospora belli, I. hominis; Dientamoeba fragilis; Insects (Onchocerca volvulus); roundworms (Ascaris lumbricoides); nematode (Necator americanis); duodenal helminths (Ancylostoma duodenale); fecal nematodes (Strongyloides stercoralis); ); Hymenolepis nana; Diphyllobothrium latum; Echinococcus granulosus, E. multilocularis; Paragonimus westermani, P. caliensis; Chlonorchis sinensis; Opisthorchis felinus, O. biberini ); Fasciola hepatica; Sarcoptes scabiei; Pediculus humanus; Phythirius pubis; and Dermatobia hominis.

  Fungi generally include, but are not limited to, Cryptococcus neoformans; Blastomyces dermatitidis; fungi (Ajellomyces dermatitidis); Histoplasma capsulatum; Coccidioides immitis; Candida albicans, C. tropicalis, C. parapsilosis, C. guilliermondii and C. krusei Candida species; A. fumigatus, Aspergillus species including yellow Aspergillus (A. flavus) and black Aspergillus (A. niger); Rhizopus species; Rhizomucor species ) Species; Cunninghammella ) Species; A. saksenaea, A. mucor and A. absidia species; Apophysomyces species; Sporothrix schenckii; Brazil Paracoccidioides brasiliensis; There are Pseudallescheria boydii; Torulopsis glabrata; and Dermatophytes species.

  Antigens that characterize autoimmune disease will typically be derived from the cell surface, cytoplasm, nucleus, mitochondria, etc. of mammalian tissue. Examples include uveitis (eg, S antigen), diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, primary myxedema, thyroid poisoning, rheumatoid arthritis, pernicious anemia , Addison's disease, scleroderma, autoimmune atrophic gastritis, premature menopause (rare case), male infertility (rare case), juvenile diabetes, Goodpascher's syndrome, pemphigus vulgaris, pemphigoid, cross Sensitive ophthalmitis, gonogenic uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis (rare case), ulcerative colitis, Sjogren's syndrome, There are antigens specific to Wegener's granulomatosis, poly / dermatomyositis and discoid lupus erythematosus.

  Antigens that are allergens are generally proteins or glycoproteins, but allergens may be low molecular weight allergenic haptens that cause allergies after covalent binding to a protein carrier (Remington's Pharmaceutical Sciences). Allergens include pollen, dust, moths, spores, dandruff, insects and antigens derived from food. Specific examples include urushiol (pentadecyl catechol or heptadecyl catechol) species such as poison ivy, poison oak and poison sumac, and ragweed and related sesquiterpenoid lactones. .

  Antigens that characterize tumor antigens will typically be derived from the cell surface, cytoplasm, nucleus, organelle, etc. of the cells of the tumor tissue. Examples include proteins encoded by mutant oncogenes; viral proteins associated with tumors; and antigens unique to tumor proteins including tumor mucins and glycolipids. Tumors include, but are not limited to, from the following cancer sites and types: lips, nasopharynx, pharynx and oral cavity, esophagus, stomach, colon, rectum, liver, gallbladder, bile duct dendritic structure , Pancreas, larynx, lungs and bronchi, skin melanoma, breast, neck, uterus (uteri, uterus), ovary, bladder, kidney, brain and other parts of the nervous system, thyroid, prostate, testis, Hodgkin's disease, There are non-Hodgkin lymphoma, multiple myeloma and leukemia. Viral proteins associated with tumors are believed to be from the above virus types. Tumor-specific antigens can be proteins that are not normally expressed by tumor progenitor cells, or can be proteins that are normally expressed in tumor progenitor cells but have tumor-specific mutations. Tumor-specific antigens can be mutated forms of common proteins with altered activity or subcellular distribution. The mutation of the gene that gives rise to the tumor antigen can be in the coding part, 5 'or 3' non-coding part, or intron of the gene, in addition to those defined above, and point mutation, frameshift, deletion , Addition, duplication, chromosomal rearrangement and the like. Those skilled in the art are familiar with the various changes in normal gene structure and expression that give rise to tumor antigens. Specific examples of tumor antigens include Ig idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 of melanoma, Pmel-17 (gp100) of melanoma, MART-1 (Melan-A of melanoma) ), Melanoma p15 protein, melanoma tyrosinase, melanoma, medullary thyroid, small cell lung cancer, colon and / or bronchial squamous cell carcinoma MAGE1, 2 and 3, bladder, melanoma, breast and squamous cell carcinoma Proteins such as BAGE of melanoma, gp75 of melanoma, oncofetal antigen of melanoma; mucl mucins of breast, pancreatic and ovarian cancer; carbohydrates / lipids such as GM2 and GD2 ganglioside of melanoma; mutant p53 of cancer And oncogenes such as colon cancer mutant ras and breast cancer HER-2 / neu primitive oncogene; and viral products such as human papillomavirus protein in cervical and esophageal squamous cell carcinoma. It is also conceivable that proteinaceous tumor antigens can be provided by HLA molecules as unique peptides derived from the entire protein. Metabolic processing of proteins that produce antigenic peptides is well known in the art, see, eg, US Pat. No. 5,342,774 (Boon et al.). Thus, the method includes the delivery of antigenic peptides and such peptides throughout the larger polypeptide or protein that yields the antigenic peptide. Supply of antigenic peptides or proteins can produce humoral or cellular immunity.

  In general, a subject can be given an effective amount of a tumor antigen and / or a peptide derived therefrom by one or more of the methods detailed below. The initial amount can be accompanied by a booster amount according to standard immunization protocols in the art. Thus, the supply of tumor antigen can stimulate the proliferation of cytolytic T lymphocytes.

  In the case of protein and peptide antigens, covalent binding to an FcRn partner is meant to include binding of a single polypeptide chain by peptide bonds. Established methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY 1989) are antigenic peptides or proteins And will be used to process DNA encoding a fusion protein consisting of an FcRn partner. This DNA is placed in an expression vector and introduced into bacterial or eukaryotic cells by established methods. The fusion protein will be purified from the cells or from the culture medium by established methods.

  When administered, the conjugates of the invention are administered in a pharmaceutically acceptable formulation. Such formulations conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and supplementary immune enhancers such as cytokines, and optionally other therapeutic agents. May be. Thus, “cocktails” including conjugates and drugs are contemplated. The drug itself can bind to the FcRn binding partner and facilitate delivery of the drug across the epithelial barrier.

  The conjugates of the invention can be administered alone (as is) or in the form of a pharmaceutically acceptable salt. When used in pharmaceuticals, the salts must be pharmaceutically acceptable, but pharmaceutically unacceptable salts can be conveniently used to produce those pharmaceutically acceptable salts; Moreover, it is not excluded from the scope of the present invention. Such pharmaceutically acceptable salts include, but are not limited to, the following acids: hydrochloric acid, chlorobrominated acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluene. Some are made from sulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Furthermore, pharmaceutically acceptable salts can be prepared as alkylin metal or alkylin earth salts such as sodium, potassium or calcium salts of carboxylic acid groups.

  Suitable buffers include acetic acid and salt (1-2% W / V); citric acid and salt 1-3% W / V; boric acid and salt (0.5-2.5% W / V); sodium bicarbonate (0.5-1.0% W / V); and phosphoric acid and salts (0.8-2% W / V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W / V); chlorobutanol (0.3-0.9% W / V); parabens (0.01-0.25% W / V) and thimerosal (0.004-0.02%). W / V).

  As used herein and more fully described below, the term “carrier” refers to one or more solid or liquid excipients, diluents suitable for administration to humans or other mammals. Or encapsulated material. A “carrier” may be a natural or synthetic organic or inorganic component that is mixed with an active ingredient to facilitate administration.

  The components of the pharmaceutical composition can be combined with the conjugates of the present invention and with each other such that there are no interactions that would substantially impair the desired drug efficiency. Components of oral drug formulations include diluents, binders, lubricants, glidants, disintegrants, colorants and flavoring agents. Encapsulated materials for the production of enteric oral formulations include cellulose acetate phthalate, poly vinyl acetate phthalate, hydroxypropylmethyl cellulose phthalate and methacrylate ester copolymers. Solid oral preparations such as capsules or tablets are preferred. Elixirs and syrups are also well known oral formulations. The components of the aerosol formulation include solubilized active ingredients, antioxidants, propellants for solvent formulations and solution formulations, and ultrafine and suspended active ingredients, dispersion aids and propellants for suspension formulations. is there. While injectable formulations must be sterile, the oral, aerosol, and nasal formulations of the present invention may not be sterile, so such formulations are distinguished from prior art injectable formulations. There is.

  The term “adjuvant” is meant to include any substance that is included or concomitantly included in a conjugate of the invention and that non-specifically enhances the subject's immune response. Adjuvants include aluminum compounds, such as gels, aluminum hydroxide and phosphate, and Freund's complete or incomplete adjuvant (where the conjugate is included in the aqueous phase of a stabilized paraffin oil-in-water emulsion). There is). Paraffin oil can be replaced with another type of oil, such as squalene or peanut oil. Other materials with adjuvant properties include BCG (attenuated Mycobacterium tuberculosis), calcium phosphate, levamisole, isoprinosine, polyanions (eg, poly A: U) reutinan, pertussis toxin, cholera toxin, lipid A, There are saponins and peptides, such as muramyl dipeptide. Rare earth salts such as lanthanum and cerium can also be used as adjuvants. The amount of adjuvant depends on the subject and the specific conjugate used and can be readily determined by one skilled in the art without undue experimentation.

  Other supplemental immune enhancing agents such as cytokines can be supplied with the conjugates of the invention. Possible cytokines are those that enhance the beneficial effects that result from administering an immunomodulator in accordance with the present invention. Cytokines are factors that support cell growth and maturation, including lymphokines. The addition of cytokines is believed to increase the cytokine activity stimulated in vivo by carrying out the method of the invention. Preferred cytokines are interleukin (IL) -1, IL-2, γ-interferon and tumor necrosis factor α. Other useful cytokines are IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13 Erythropoietin, leukemia inhibitory factor, oncostatin-M, ciliary neurotrophic factor, growth hormone, prolactin, CD40-ligand, CD27-ligand, CD30-ligand, α-interferon, β-interferon and tumor necrosis factor β it is conceivable that. Other cytokines known to modulate T cell activity as deemed useful by the present invention are granulocyte and / or macrophage stimulating factors (GM-CSF, G-CSF and CSF-1), And colony-stimulating factors and growth factors including platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, transforming growth factor and fibroblast growth factor. The selection of a particular cytokine will depend on the specific regulation of the desired immune system. The activity of cytokines against specific cell types is known to those skilled in the art.

  The exact amount of the aforementioned cytokines used in the present invention will depend on various factors, including the selected conjugate, the selected dosage and timing, the mode of administration and the characteristics of the subject. The exact amount selected can be determined without undue experimentation, particularly because the limiting amount is any amount that will promote the desired immune response. Thus, depending on the delivery mode, nanogram to milligram quantities are considered useful, but nanogram to milligram quantities are considered most useful because the physiological concentrations of cytokines are similarly low.

  The formulations of the present invention are administered in an effective amount. An effective amount is the amount of conjugate that will stimulate the immune response as desired, either alone or in combination with additional amounts. This may be involved in humoral antibody responses that cause increased antibody titers in serum, improved mucosal immunity, increased clonal cytotoxic T lymphocytes, or stimulation of tolerance to antigens, including self-antigens. it can. Doses ranging from 1 nanogram / kilogram to 100 mg / kilogram are considered effective depending on the mode of administration. The preferred range is believed to be from about 500 nanograms to 500 micrograms / kilogram, most preferably from 1 microgram to 100 micrograms / kilogram. Its absolute amount will depend on the individual conjugate, including the selected conjugate, the desired immunomodulation, whether it is a single or multiple dose, and age, physical symptoms, physique and weight. It will depend on various factors, including parameters. When determining the amount of conjugate to administer for the treatment of a subject with a tumor, the size, type, location and metastasis of the tumor can be factors. These factors are well known to those skilled in the art and can only be shown by routine experimentation.

  A variety of administration routes are available. The particular mode chosen will, of course, depend on the particular conjugate chosen, the particular condition being treated, and the dosage required for therapeutic efficacy. The method of the present invention generally relates to providing a conjugate of the present invention to an epithelial surface. Preferred modes of administration are oral, pulmonary, intrabile and intranasal.

  The composition can conveniently be given in unit dosage form and can be prepared by any method known in the pharmacy art. Any method includes the step of bringing the conjugate into association with a carrier which constitutes one or more accessory ingredients. Generally, the composition is made by shaping the product, if necessary, after the conjugate is uniformly and tightly combined with the liquid carrier, the finely divided solid carrier or both.

  Other delivery systems can include time release, delayed release or sustained release delivery systems. Such a system can avoid repeated administrations of the conjugates of the invention and is increasingly advantageous for the subject and the physician. Many types of release / delivery systems are available and known to those skilled in the art. They include polymer base systems such as polyacetic acid, polyglycolic acid, polyanhydrides and polycaprolactone; compressed tablets using wax coatings, conventional binders and excipients. Bioadhesive polymer systems that facilitate the supply of substances to the intestinal epithelium are known and described in published PCT application WO 93/21906. Capsules that deliver drugs to the intestinal epithelium are also described in published PCT application WO 93/19660.

Example
material
Abbreviation
BSA, bovine serum albumin; cDNA, complementary deoxyribonucleic acid; CT-B, cholera toxin B subunit; DMEM, Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; DOC, desoxycholate; ECL, enhanced chemiluminescence; Solvent assay; Hank's balanced salt solution without HBSS, calcium or magnesium; HEPES, N- [2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid]; hGH, human growth hormone; IEC, intestinal epithelium cells; KI, potassium iodide; MHC, major histocompatibility complex; NaOH, sodium hydroxide; NH ₄ Cl, ammonium chloride; NHS-rhodamine, N- hydroxysuccinimidyl - rhodamine; RNA-ribonucleic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; SATA, N-succinimidyl S-acetylthioacetate; SDS-PAGE, sodium dodecyl sulfate-po Acrylamide gel electrophoresis; sulfo-LC-SPDP, sulfosuccinimidyl 6- [3- (2-pyridylthio) propionamide] hexanoate; sulfo-NHS-biotin, sulfosuccinimide biotin; sulfo-SMCC, sulfosucciniimi Zir 4- (N-maleimidomethyl) cyclohexane-1-carboxylate.

chemicals
The cDNA cycle kit was purchased from Invitrogen (San Diego, Calif.). Taq polymerase was purchased from Perkin-Elmer Cetus (Norwalk, CT). The Circum Vent ^™ kit was purchased from New England Biolabs (Beverly, Mass.). Radionuclides and chemicals were purchased from DuPont / NEN (Boston, MA). HBSS and DMEM were purchased from GIBCO / Life Technologies (Gaithersburg, MD). RPMI 1640 was purchased from Cellgro (Herndon, VA). L-glutamine was purchased from Sergro. Protein A-Sepharose was purchased from Pharmacia Biotech (Piscataway, NJ). Streptavidin-horseradish peroxidase, sulfo-LC-SPDP, sulfo-NHS-biotin, sulfo-SMCC, SATA and immobilized ficin were purchased from Pierce (Rockford, IL). Balb / c mice were purchased from Charles River Laboratories (Wilmington, Mass.). The ECL kit was purchased from Amersham (Arlington Heights, IL). Plasmin, AvidChrom-Protein A, Protein G-Sepharose, BSA, Cholera Toxin B subunit, anti-hGH antibody and all other chemicals were purchased from Sigma (St. Louis, MO).

Example 1: Expression of FcRn mRNA in human intestinal epithelial primary cells and cell lines Total RNA was extracted from adult intestinal cells by standard methods well known in the art (Sambrook et al., Supra). A cDNA substrate for reverse transcriptase-polymerase chain reaction (RT-PCR) using a cDNA cycle kit (Invitrogen, San Diego, CA) was prepared using 1 microgram of RNA from each cell type as a template. . The cDNA was subjected to 30 PCR cycles with primers TGCTGGGCTGTGAACTG and CGCTTTTAGCAGTCGGAA using Taq polymerase (Perkin Elmer Citas, Norwalk, CT) according to the manufacturer's instructions. The PCR cycle conditions were denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 2 minutes and extension at 72 ° C. for 3 minutes. Amplified products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining, which was expected to be approximately 800 base pair amplified products in all samples except adult colonic epithelial cells. Showed existence. To confirm the identity of the RT-PCR amplification product, the DNA band was excised from the agarose gel, subcloned into pCR II (Invitrogen, San Diego, Calif.), And Prism ^™ dye-deoxy terminator cycle sequencing The kits (Applied Biosystems, Foster City, Calif.) Were used to sequence using primers from both the vector and human FcRn sequences. The reaction product was analyzed on an Applied Biosystems sequencer. The sequence of the amplified product matched exactly with the FcRn gene sequence, confirming the identity of the expressed gene.

Example 2: Detection of FcRn mRNA by Northern blot To confirm the expression of FcRn in human intestinal epithelial cells and cell lines, using an RNA sample prepared as described in Example 1, from adult intestinal cells, Northern blots were also made from two human adenocarcinoma cell lines CaCO-2 and HT-29 originating from the colon. The RNA samples were separated by formaldehyde / agarose gel electrophoresis and nylon by standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY 1989). Transferred to the membrane. The membrane was probed from the 3 ′ untranslated portion of FcRn by standard methods using a 120 base pair probe radiolabeled with ³² P. Northern blot autoradiograms demonstrated the presence of 1.5 kilobase hFcRn transcripts in enterocytes and both cell lines. Thus, the expression of FcRn in adult intestinal epithelial cells and cell lines was demonstrated by two different RNA detection methods.

Example 3: Labeling and immunoprecipitation of MHC class I-related Fc receptor (FcRn) from intestinal epithelial cells The expression of FcRn in human intestinal epithelial cells was confirmed by protein immunoprecipitation. Caco-2 cells are metabolically labeled with ³⁵ S-methionine (DuPont / NEN, Boston, Mass.) And the protein is purified by methods well known in the art (Harlow and Lane, Antibodies : A Laboratory Manual). FcRn was immunoprecipitated from cell extracts using standard methods using a polyclonal rabbit anti-rat MHC class I-related FcR heavy chain specific antiserum conjugated to protein A-sepharose (FcRn is a Simister) And purified by well-established methods of Ress, 1985, European J. Immunology, 15: 733-8, and can be used to collect serum after immunizing rats, Harlow and Lane, above). Immunoprecipitates were separated by SDS-PAGE and visualized by autoradiography. A 48 kilodalton FcRn protein was found, confirming the expression seen at that RNA concentration.

Example 4: Expression of FcRn protein on the cell surface of human intestinal epithelial cells
Approximately 3X10 ⁷ HT-29 intestinal epidermis cells were isolated from tissue culture plates by non-enzymatic methods and ice-cold Hanks balanced salt solution without calcium or magnesium (HBSS-, GIBCO / Life Technologies, Gaithersburg) , MD) and washed 4 times. To label cell surface proteins, the washed cells were incubated twice for 20 minutes with 1.5 ml of 0.5 mg / ml sulfo-NHS-biotin (Pierce, Rockford, IL) in DMSO. Labeled cells are washed 5 times with 50 mM NH ₄ Cl and incubated for 20 minutes with 10 ml RPMI 1640 (Sergro, City, USA) containing 1 mM L-glutamine (Mediatech, Washington, DC) , And washed 4 times with HBSS-. The cells were lysed and then precleared overnight using protein A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ) using standard techniques well known in the art. SDS and desoxycholic acid (DOC) were added to the supernatant to a final concentration of 0.1% and 0.5%, respectively. Lysates were precleared with normal rabbit serum and immunoprecipitated with polyclonal rabbit anti-rat MHC class I related FcR antibodies by methods well known in the art. Immunoprecipitates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Nitrocellulose membranes were processed for incubation with streptavidin-horseradish peroxidase (Pierce, Rockford, IL) diluted 1: 10,000 as suggested by the manufacturer. The membrane was then processed for detection of bound horseradish peroxidase using an ECL kit (Amersham, Arlington Heights, IL). Luminescence due to cleavage of the chemiluminescent substrate was detected by exposure of the membrane to a photosensitive film. Film exposure showed that FcRn was expressed on the surface of HT-29 intestinal epithelial cells.

Example 5: Functional activity of human FcRn on the cell surface of intestinal epithelial cells To demonstrate that FcRn expressed on the cell surface of intestinal epithelial cells was functional, Caco-2 cells and adult jejunal intestinal epithelium Cells (IEC) were examined for their ability to bind Fc fragments of antibodies. Caco-2 and jejunum IEC were dispensed into microcentrifuge tubes (2 × 10 ⁶ cells / tube) and pelleted at 2000 rpm at 4 ° C. for 2-3 minutes. The cell pellet was washed once at 4 ° C. in DMEM, pH 6.0 or pH 8.0 containing 20 mM HEPES and resuspended in 0.2 ml of the same culture. The cell suspension was transferred to a 12-well plate for assay. ¹²⁵ I-Fc fragment (200 ng / ml, 4 × 10 ⁻⁹ M) in DMEM, pH 6.0 or pH 8.0 containing 20 mM HEPES, 1.0 mM KI and 0.1% fish gelatin was added to unlabeled human IgG (3.3 × 10 ^{− 6} M) was added to each well with or without 0.5 mg / ml. Cells were allowed to bind IgG or Fc for 2 hours at 37 ° C. in a humidified atmosphere of 5% CO ₂ . Cells were transferred to a microcentrifuge tube and pelleted at 2000 rpm at 4 ° C. for 2-3 minutes. Unbound ¹²⁵ I-Fc was removed by washing the cell pellet once with cold DMEM, pH 6.0 or pH 8.0 containing 20 mM HEPES at 4 ° C. Cells were disrupted in 0.5 ml 0.1 M NaOH and the resulting solution was transferred to a scintillation vial. ¹²⁵ I was quantified using a CliniGamma 1272 γ counter (LKB Wallac, Piscataway, NJ). Both Caco-2 cells and adult jejunum IEC specifically bind ¹²⁵ I-Fc at pH 6.0 instead of pH 8.0 and are functional pH dependent as seen for rat neonatal FcRn and cloned human FcRn Binding has been demonstrated (Suetry et al., J. Exp. Med., 180: 2377-2381; December 1994).

Example 6 Production of Human Immunoglobulin G Non-specifically purified immunoglobulin G from human, mouse, rat, goat, pig, cow and other species was obtained from Sigma Chemical Company, Pierce Chemical, High Clone Laboratories. (HyClone Laboratories), ICN Biomedicals and Organon Teknika-Cappel.

  Immunoglobulin G can also be isolated by serum ammonium sulfate precipitation. The protein precipitate is further fractionated by ion exchange chromatography or gel filtration chromatography to isolate substantially purified non-specific IgG. Non-specific IgG means that there is no single specificity that is dominant in the antibody population or pool.

  Immunoglobulin G can also be purified from serum by adsorption to protein A bound to a solid support such as Protein A-Sepharose (Pharmacia), AvidChrom-Protein A (Sigma) or Protein G-Sepharose (Sigma) . Other purification methods of IgG are well known to those skilled in the art and can be used for the isolation of non-specific IgG.

Example 7: Production of human immunoglobulin G Fc fragment To produce the Fc fragment of human IgG, the IgG isolated as in Example 6 was transferred to its manufacturer using immobilized papain (Pierce). Digest according to the protocol provided. Other proteases, such as plasmin (Sigma) or immobilized ficin (Pierce), that digest IgG to produce a complete Fc fragment that can bind to the Fc receptor are known to those skilled in the art and produce Fc fragments Can be used. The digested immunoglobulin is then incubated with an affinity substrate such as protein A-sepharose or protein G-sepharose. The unbound portion of the IgG is eluted from the affinity substrate by extensive washing in batch or column format. The IgG Fc fragment is then eluted by the addition of a buffer that is incompatible with Fc adsorption binding. Other methods effective in the purification of Fc fragments can also be used.

Example 8: Binding of compounds to human immunoglobulin Fc fragments
In order to supply compounds by FcRn transport mechanism, such compounds can be bound to intact IgG or Fc fragments. Cross-linking chemistry and reagents useful for such purposes are well known in the art. The nature of the cross-linking reagent used to bind the complete IgG or Fc fragment and the supplied compound is not limited by the present invention. Any cross-linking reagent can be used as long as (a) the activity of the compound is retained, and (b) the binding of the Fc portion of the conjugate by FcRn is not adversely affected.

An example of effective one-step cross-linking of Fc and compounds is the use of Fc in which sodium periodate is used in sodium phosphate buffer for 30 minutes at room temperature followed by overnight incubation at 4 ° C. with the bound compound. It is oxidation. Coupling is performed by derivatizing both the compound and the Fc fragment with sulfosuccinimidyl 6- [3- (2-pyridylthio) propionamido] hexanoate (sulfo-LC-SPDP, Pierce) for 18 hours at room temperature. You can also. Coupling can also be performed by derivatizing the Fc fragment and the desired compound supplied with various cross-linking reagents that will subsequently form covalent bonds. An example of this reaction is derivatization of an Fc fragment with sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC, Pierce), where the compound bound to Fc is Thiolated with N-succinimidyl S-acetylthioacetate (SATA). These derivative components are purified without a crosslinker and mixed to crosslink for 1 hour at room temperature. Other cross-linking reagents including aldehyde, imide, cyano, halogen, carboxyl, activated carboxyl, anhydride and maleimide functional groups are known to those skilled in the art and can also be used to attach compounds to Fc fragments. The choice of cross-linking reagent will of course depend on the nature of the compound that is desired to be bound to Fc. The above-mentioned cross-linking reagent is effective for protein-protein binding. Heterobifunctional cross-linking reagents such as ABH, M ₂ C ₂ H, MPBH and PDPH are useful for conjugation with proteinaceous FcRn binding molecules when the compound to be bound is a carbohydrate or has a carbohydrate residue (Pierce Chemical Company, Rockford, IL). Another method for combining proteins and carbohydrates is disclosed by Brumeanu et al. (Genetic Engineering News, Oct. 1, 1995, page 16). If the compound to be bound is a lipid or has a lipid residue, which is convenient as a binding site to the FcRn binding molecule, crosslinkers such as SPDP, SMPB and their derivatives can be used (Pierce).・ Chemical Company, Rockford, IL). It is also possible to bind any molecule that should be supplied by non-covalent means. One convenient way to achieve non-covalent binding is to generate antibodies, such as monoclonal antibodies, against the supplied compound by methods well known in the art and the correct Fc portion and the desired antigen. It is to select monoclonal antibodies having binding properties. Next, the supplied antigen is pre-bound to the monoclonal antibody carrier. In any of the above cross-linking reactions, it is important to purify the derivative compound without including a cross-linking reagent. It is also important to purify the final conjugate substantially free of unbound reactants. Purification can be achieved by affinity chromatography, gel filtration chromatography or ion exchange chromatography based on the nature of each component of the conjugate. A particularly preferred method is to first perform an affinity purification step that retains Fc and Fc compound conjugates using protein A-sepharose, followed by gel filtration or ion exchange chromatography based on the mass, size or charge of the Fc conjugate. Do the graphic. The first step of this purification scheme ensures that the conjugate binds to FcRn, which is an essential requirement of the present invention.

Example 9: Supply of foreign antigens assisted by IgG across the intestinal epithelial barrier To examine the ability of Fc binding partner-antigen conjugates to be transported across the epithelial barrier, foreign antigens for administration to mice Bind to IgG molecule. A convenient foreign antigen is the fluorescent dye rhodamine because it can be visualized in frozen semi-thin sections of the intestinal epithelium. Non-specific mouse IgG, cholera toxin B subunit produced as described in Example 6 by incubation as presented by the manufacturer using succinyl rhodamine (Molecular Probes, Eugene, OR). Rhodamine is covalently linked to (Sigma) and ovalbumin (Sigma). The IgG-rhodamine conjugate is purified by protein G-Sepharose affinity chromatography. After dialysis to remove unbound succinyl rhodamine, cholera toxin B (CT-B) -rhodamine and ovalbumin-rhodamine conjugate are purified by gel filtration or ion exchange chromatography. The presence of the conjugate is assayed by measuring the fluorescence of the eluate fraction. Functional binding of IgG and CT-B subunit conjugates can be examined by binding to FcRn and ganglioside GMI, respectively. Cholera toxin B-rhodamine and ovalbumin-rhodamine serve as positive and negative controls, respectively.

Balb / c mice received 75 nanomolar NaHCO ₃ to prevent gastric degradation with or without 0.2 nanomolar of the three rhodamine conjugates with or without 0.2 nanomolar unlabeled cholera toxin as a non-specific adjuvant. And administered by intragastric administration in the presence of 20 mg / ml soybean trypsin inhibitor. After 6 hours, the mice are sacrificed and the intestine is removed, frozen, and processed for semi-thin sections. Intestinal epithelial sections were illuminated with a fluorescence microscope and examined for intracellular fluorescence. The presence of fluorescence in the intestinal epithelial cells of mice given IgG-rhodamine indicates that the IgG conjugate is effectively transported across the intestinal epithelial barrier toward the basolateral direction at the tip. FcRn can transport the immunogen as a conjugate with the FcRn binding partner.

Example 10: Transgenic homozygous for deletion of mouse mucosal immune response β ₂ -microglobulin (a critical component of Fc receptor function) to antigen-IgG conjugates orally delivered by FcRn-dependent cell permeation Mice and their normal wild type littermates are used to study mucosal immune response development. If rhodamine-IgG causes a mucosal immune response by binding to the apical membrane Fc receptor, a positive immune response should be found in the wild-type rather than in the β ₂ -microglobulin “knockout” mouse. In contrast, rhodamine-cholera toxin B subunit (CT-B) is both in wild-type and “knockout” mice, as CT-B cell penetration across the epithelial barrier does not depend on binding to the apical membrane Fc receptor. Should cause a positive immune response. Rhodamine-ovalbumin does not enter cell-permeable vesicles (although it can enter the intestinal epithelium by liquid phase endocytosis) and should not elicit an immune response in any mouse.

Three groups of wild type and β ₂ -microglobulin knockout mice are immunized orally with the three rhodamine conjugates described in Example 9. Parallel experiments are performed with 0.2 nanomolar cholera toxin as a non-specific adjuvant. An equimolar amount of rhodamine conjugate is administered intragastrically. Mice are “immunized” a total of 3 times every 10 days by this method. Two weeks after the third oral immunization, mice are sacrificed and rhodamine-specific immune responses are confirmed by standard methods for intestinal secretion and serum by ELISA. Anti-rhodamine serum immunoglobulin is most evident in wild-type mice given CT-B and IgG rhodamine conjugates. Knockout mice lacking β ₂ -microglobulin develop mucosal immune responses against rhodamine-CT-B but not rhodamine-IgG, and receptor-dependent cell penetration plays an essential role in mucosal immune responses It is shown that it plays. The control rhodamine-ovalbumin conjugate elicits little or no immune response in either wild type or β ₂ -microglobulin knockout mice.

  Those skilled in the art will understand or be able to ascertain using no more than routine experimentation many equivalents to the unique products and processes described above. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

Claims (25)

A method of modulating the immune system of a mammal, comprising administering to the mammalian epithelial barrier in need of such immunomodulation an effective amount of a conjugate of an antigen and an FcRn binding partner, wherein Wherein the antigen is
An antigen characteristic of the pathogen,
Antigens characteristic of autoimmune diseases,
The above method, wherein the antigen is an allergen characteristic antigen or an antigen characteristic of a tumor.   2. The method of claim 1, wherein the FcRn binding partner is non-specific IgG or an FcRn binding fragment of IgG.   3. The method according to claims 1-2, wherein the conjugate is administered orally to the intestinal epithelium or to the lung by aerosol.   4. A method according to claims 1-3, wherein the antigen exhibits pathogenic characteristics.   4. A method according to claims 1 to 3 wherein the antigen exhibits characteristics of an autoimmune disease.   4. The method of claims 1-3, wherein the antigen exhibits tumor characteristics.   4. A method according to claims 1-3, wherein the antigen exhibits allergen characteristics.   8. The method of claims 1-7, wherein the antigen is covalently bound to the FcRn binding partner.   The method according to claims 1 to 8, wherein the FcRn binding partner is an Fc fragment of IgG. A formulation formulated for medical use,
A conjugate of an antigen and an FcRn binding partner, wherein the antigen exhibits characteristics of a pathogen, autoimmune disease, allergen or tumor, and comprises a pharmaceutically acceptable carrier.   11. The formulation of claim 10, wherein the antigen exhibits pathogenic characteristics.   11. The preparation according to claim 10, wherein the antigen exhibits characteristics of an autoimmune disease.   11. The formulation according to claim 10, wherein the antigen exhibits allergen characteristics.   11. The preparation according to claim 10, wherein the antigen exhibits tumor characteristics.   The preparation according to claims 10 to 14, wherein the FcRn binding partner is non-specific IgG or an FcRn-binding fragment of IgG.   The preparation according to claims 10 to 14, wherein the FcRn binding partner is an Fc fragment of IgG.   17. A formulation according to claims 10-16, wherein the antigen is covalently bound to an FcRn binding partner.   18. A formulation according to claims 10-17, wherein the formulation is a unit dose containing an effective amount of a conjugate that modulates the immune system, the unit dose being constructed and adjusted for delivery to the epithelial barrier.   Use of a conjugate of an antigen and an FcRn binding partner in the manufacture of a medicament for modulating the immune response of a mammal, wherein the antigen is an antigen that is characteristic of a pathogen, autoimmune disease, allergen or tumor .   20. The use according to claim 19, wherein the FcRn binding partner is non-specific IgG or an FcRn binding fragment of IgG.   20. Use according to claim 19, wherein the FcRn binding partner is an Fc fragment of IgG.   Use according to claims 19 to 21, wherein the formulation is an oral formulation.   Use according to claims 19 to 21, wherein the formulation is an aerosol formulation.   Use according to claims 19 to 21, wherein the formulation is a nasal formulation.   Use according to claims 19 to 21, wherein the formulation is not sterile.